Aspergillus oryzae 5-aminolevulinic acid synthases and nucleic acids encoding same

ABSTRACT

The present invention relates to Aspergillus oryzae 5-aminolevulinic acid synthases and isolated nucleic acid fragments comprising nucleic acid sequences encoding the 5-aminolevulinic acid synthases as well as nucleic acid constructs, vectors, and recombinant host cells comprising the nucleic acid sequences. The invention also relates to methods of producing the 5-aminolevulinic acid synthases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 08/871,266filed Jun. 9, 1997 which is a continuation-in-part of application Ser.No. 60/019,399 filed Jun. 10, 1996, the contents of which are fullyincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to Aspergillus oryzae 5-aminolevulinicacid synthases and isolated nucleic acid fragments comprising nucleicacid sequences encoding the 5-aminolevulinic acid syntheses. Theinvention also relates to nucleic acid constructs, vectors, and hostcells comprising the nucleic acid sequences as well as methods forproducing the 5-aminolevulinic acid synthases.

2. Description of the Related Art

Heme, a chelate complex of protoporphyrin IX and iron, serves as aprosthetic group of hemoproteins. Protoporphyrin IX consists of aporphyrin ring, substituted with four methyl groups, two vinyl groups,and two propionic acid groups, which acquires an iron atom to form heme.The biosynthesis of heme from glycine and succinyl-CoA involves eightenzymatic steps. The first enzyme in the biosynthetic pathway is5-aminolevulinic acid synthase which catalyzes the condensation ofglycine and succinyl-CoA to form 5-aminolevulinic acid. In thebiosynthesis of heme in liver cells and differentiating erythrocytes,5-aminolevulinic acid synthase is a key regulatory enzyme.

The conversion of an apoprotein into a hemoprotein depends on theavailability of heme provided by the heme biosynthetic pathway. Theapoprotein form of the hemoprotein combines with heme to produce theactive hemoprotein. The active hemoprotein acquires a conformation whichmakes the hemoprotein more stable than the apoprotein to proteolyticattack. If the amount of heme produced by a microorganism is lessrelative to the amount of the apoprotein produced, the apoprotein willaccumulate and undergo proteolytic degradation lowering the yield of theactive hemoprotein.

In order to overcome this problem, Jensen showed that the addition ofheme or a heme-containing material to a fermentation medium led to asignificant increase in the yield of a peroxidase produced byAspergillus oryzae (WO 93/19195). While heme supplementation of afermentation medium results in a significant improvement in the yield ofa hemoprotein, it is non-kosher, costly, and difficult to implement on alarge scale.

The cloning and sequencing of a 5-aminolevulinic acid synthase gene fromAspergillus nidulans (Bradshaw et al., 1993, Current Genetics2233:501-507) have been disclosed.

It is an object of the present invention to provide new 5-aminolevulinicacid synthases and genes encoding same.

SUMMARY OF THE INVENTION

The present invention relates to substantially pure 5-aminolevulinicacid synthases obtained from Aspergillus oryzae and to isolated nucleicacid fragments comprising a nucleic acid sequence which encodes anAspergillus oryzae 5-aminolevulinic acid synthase. The present inventionfurther provides nucleic acid constructs, vectors, and recombinant hostcells comprising a nucleic acid fragment of the present invention aswell as methods for producing the 5-aminolevulinic acid synthases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of plasmid pSE04.

FIG. 2 shows a restriction map of a 4.2 kb genomic fragment containingan Aspergillus oryzae 5-aminolevulinic acid synthase gene. Scale inkilobases (kb) is shown under the map. The arrow represents the locationof the open reading frame of the gene.

FIGS. 3A-3B shows the nucleotide and deduced amino acid sequences of anAspergillus oryzae 5-aminolevulinic acid synthase gene (SEQ ID NOS: 1and 2, respectively). Potentially important transcriptional sites, CCAATbox and TATA box are underlined. The two conserved putative HRM motifsare boxed; the glycine loop involved in pyridoxal phosphate co-factorbinding is circled and the important lysine is indicated with anasterisk.

FIG. 4 shows the conserved heme regulatory motifs in various5-aminolevulinic acid synthase genes. The pentapeptide motifs are boxed.

FIGS. 5A-5C shows the alignment of the deduced amino acid sequences for5-aminolevulinic acid synthases from Aspergillus oryzae, Aspergillusnidulans, Saccharomyces cerevisiae and human erythroid (SEQ ID NOS:2,16, 17 and 18, respectively). Conserved amino acids are boxed.

FIG. 6 shows a restriction map of plasmid pBANe6.

FIG. 7 shows a restriction map of plasmid pSE31.

FIG. 8 shows the construction of plasmid pJVi9.

FIG. 9 shows a restriction map of plasmid pJeRS6.

FIG. 10 shows a restriction map of plasmid pJRoC50.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, as mentioned above, relates to 5-aminolevulinicacid synthases obtained from an Aspergillus oryzae strain. Strains ofthis species are readily accessible to the public in a number of culturecollections, such as the American Type Culture Collection (ATCC),Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM),Centraalbureau Voor Schimmelcultures (CBS), International MycologicalInstitute (IMI), Agricultural Research Service Patent CultureCollection, Northern Regional Research Center (NRRL), and Institute forFermentation in Osaka, Japan (IFO).

In a preferred embodiment, the present invention relates to5-aminolevulinic acid synthases obtained from Aspergillus oryzae or amutant strain thereof. In a more preferred embodiment, the presentinvention relates to 5-aminolevulinic acid synthases obtained fromAspergillus oryzae IFO 4177 or a mutant strain thereof, e.g., the5-aminolevulinic acid synthase having the amino acid sequence set forthin SEQ ID NO:2.

The present invention also relates to 5-aminolevulinic acid synthaseswhich are encoded by nucleic acid sequences which are capable ofhybridizing under high stringency conditions (i.e., prehybridization andhybridization at 45° C. in 5 X SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and 50% formamide) with a probe whichhybridizes with the nucleic acid sequence set forth in SEQ ID NO:1 underthe same conditions. The gene, or an oligonucleotide based thereon, canbe used as a probe in Southern hybridization to isolate homologous genesof any Aspergillus species. In particular, such probes can be used forhybridization with the genomic or cDNA of the species of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding 5-aminolevulinic acid synthase genetherein. Degenerate PCR primers (oligonucleotides) can be used withgenomic DNA or cDNA segments to amplify 5-aminolevulinic acidsynthase-specific gene segments.

Identification and isolation of 5-aminolevulinic acid synthase genesfrom a source other than those specifically exemplified herein can beachieved by utilization of the methodology described in the presentexamples, with publicly available Aspergillus strains.

For purposes of the present invention, the term "obtained from" meansthat the 5-aminolevulinic acid synthase is produced by a specificsource, e.g., an Aspergillus strain, or by a cell in which a gene fromthe source encoding the 5-aminolevulinic acid synthase has beeninserted.

The invention also encompasses 5-aminolevulinic acid synthase variantswhich have at least about 80%, preferably about 85%, more preferablyabout 90%, and most preferably about 95% homology with the amino acidsequence set forth in SEQ ID NO:2, and which qualitatively retains theactivity of the 5-aminolevulinic acid synthases described herein. Thepresent invention is also directed to 5-aminolevulinic acid synthasevariants which have an amino acid sequence which differs by three aminoacids, preferably by two amino acids, and more preferably by one aminoacid from the amino acid sequence set forth in SEQ ID NO:2. Eachdifference may be an insertion or deletion of an amino acid or thesubstitution of an amino acid residue by a different amino acid. Usefulvariants within the categories defined above include, for example, onesin which conservative amino acid substitutions have been made, whichsubstitutions do not significantly affect the activity of the protein.By conservative substitution is meant that amino acids of the same classmay be substituted by any other amino acid of that class. For example,the nonpolar aliphatic residues Ala, Val, Leu, and Ile may beinterchanged, as may be the basic residues Lys and Arg, or the acidicresidues Asp and Glu. Similarly, Ser and Thr are conservativesubstitutions for each other, as are Asn and Gln.

The physical-chemical properties of the 5-aminolevulinic acid synthasesof the present invention may be determined using various techniques wellknown in the art including, but not limited to, SDS-PAGE, isoelectricfocusing, and cross-reaction irmunoidentity tests. The 5-aminolevulinicacid synthases of the present invention may be assayed using methodsknown in the art.

The 5-aminolevulinic acid synthases of the present invention may bepurified by a variety of procedures known in the art including, but notlimited to, chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures(e.g.., preparative isoelectric focusing), differential solubility(e.g., ammonium sulfate precipitation), or extraction (see, for example,Protein Purzfication, eds. J.-C. Janson and Lars Ryden, VCH Publishers,New York, 1989). As defined herein, a "substantially pure"5-aminolevulinic acid synthase is a 5-aminolevulinic acid synthase whichis essentially free of other non-5-aminolevulinic acid synthaseproteins, for example, at least about 20% pure, preferably about 40%pure, more preferably about 60% pure, even more preferably about 80%pure, most preferably about 90% pure, and even most preferably at leastabout 95% pure, as determined by SDS-PAGE.

The present invention also relates to nucleic acid fragments comprisinga nucleic acid sequence which encodes a 5-aminolevulinic acid synthaseof the present invention and to nucleic acid constructs comprising anucleic acid fragment of the present invention.

In a preferred embodiment, the nucleic acid sequence encodes a5-aminolevulinic acid synthase obtained from Aspergillus oryzae. In amore preferred embodiment, the nucleic acid sequence encodes a5-aminolevulinic acid synthase obtained from Aspergillus oryzae IFO4177, e.g., the nucleic acid sequence set forth in SEQ ID NO:1. Thepresent invention also encompasses nucleic acid sequences which encode a5-aminolevulinic acid synthase having the amino acid sequence set forthin SEQ ID NO:2, which differ from SEQ ID NO:1 by virtue of thedegeneracy of the genetic code. The nucleic acid sequences of thepresent invention encompass both the genomic sequence depicted thereinas well as the corresponding cDNA and RNA sequences, and the phrase"nucleic acid sequence" as used herein will be understood to encompassall such variations including synthetic DNA.

The present invention also relates to nucleic acid constructs comprisinga nucleic acid fragment of the invention. "Nucleic acid construct" shallgenerally be understood to mean a nucleic acid molecule, either single-or double-stranded, which is isolated from a naturally occurring gene orwhich has been modified to contain segments of nucleic acid which arecombined and juxtaposed in a manner which would not otherwise exist innature. In a preferred embodiment, the nucleic acid constructs areoperably linked to regulatory regions capable of directing theexpression of the 5-aminolevulinic acid synthase in a suitableexpression host.

The present invention also provides recombinant vectors comprising anucleic acid construct of the present invention. In a preferredembodiment, the nucleic acid sequence is operably linked to a promotersequence. In another preferred embodiment, the vectors of the presentinvention further comprise a transcription termination signal and/or aselectable marker.

The recombinant vectors of the invention are useful for the expressionof an Aspergillus oryzae 5-aminolevulinic acid synthase gene in activeform. A useful vector contains an element that permits stableintegration of the vector into the host cell genome or autonomousreplication of the vector in a host cell independent of the genome ofthe host cell, and preferably one or more phenotypic markers whichpermit easy selection of transformed host cells. The vector may alsoinclude control sequences such as a promoter, ribosome binding site,translation initiation signal, and, optionally, a selectable marker orvarious activator or repressor sequences. To permit the secretion of theexpressed protein, nucleic acids encoding a signal sequence may beinserted prior to the coding sequence of the gene. For expression underthe direction of control sequences, a 5-aminolevulinic acid synthasegene to be used according to the present invention is operably linked tothe control sequences in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences.

The vectors carrying a nucleic acid construct of the present inventionmay be any vector which can conveniently be subjected to recombinant DNAprocedures. The choice of a vector will typically depend on the hostcell into which the vector is to be introduced. The vector may be anautonomously replicating vector, i.e., a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome. Alternatively, the vectormay be one which, when introduced into a host cell, is integrated intothe host cell genome and replicated together with the chromosome(s) intowhich it has been integrated. The vector system may be a single vectoror plasmid or two or more vectors or plasmids which together contain thetotal DNA to be integrated into the genome.

In the vectors, the DNA sequence should be operably linked to a suitablepromoter sequence. The promoter may be any DNA sequence which showstranscriptional activity in the host cell of choice and may be obtainedfrom genes encoding proteins either homologous or heterologous to thehost cell. Examples of suitable promoters for directing thetranscription of the nucleic acid construct of the invention, especiallyin a bacterial host, are the promoter of the lac operon of E. coli, theStreptomyces coelicolor agarase gene dagA promoters, the promoters ofthe Bacillus licheniformis α-amylase gene (amyL), the promoters of theBacillus stearothermophilus maltogenic amylase gene (amyM), thepromoters of the Bacillus amyloliquefaciens α-amylase (amyQ), thepromoters of the Bacillus subtilis xylA and xylB genes, the prokaryoticβ-lactamase promoter (Villa-Kamaroff et al., 1978, Proceedings of theNational Academy of Sciences U.S.A. 75:3727-3731) or the tac promoter(DeBoer et al., 1983, Proceedings of the National Academy of SciencesU.S.A. 80:21-25). Further promoters are described in "Useful proteinsfrom recombinant bacteria" in Scientific American, 1980, 242:74-94; andin Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d ed., ColdSpring Harbor, N.Y., 1989. In a yeast host, a useful promoter is theeno-l promoter. For transcription in a fungal host, examples of usefulpromoters are those obtained from the genes encoding Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral α-amylase, Aspergillus niger acid stable α-amylase, Aspergillusniger or Aspergillus awamori glucoamyIase (glaA), Rhizomucor mieheilipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase or Aspergillus nidulans acetamidase. Particularlypreferred promoters are the TAKA amylase, NA2-tpi (a hybrid of thepromoters from the genes encoding Aspergillus niger neutral α-amylaseand Aspergillus oryzae triose phosphate isomerase), and glaA promoters.

The vectors of the invention may also comprise a suitable transcriptionterminator and, in eukaryotes, polyadenylation sequences operablyconnected to the DNA sequence encoding a 5-aminolevulinic acid synthaseof the present invention. Termination and polyadenylation sequences maybe obtained from the same sources as the promoter. The vectors mayfurther comprise a DNA sequence enabling the vectors to replicate in thehost cell in question. Examples of such sequences are the origins ofreplication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1, andpIJ702.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophsand the like. The selectable marker may be selected from the groupconsisting of, but not limited to, amdS, pyrG, argB, niaD, sC, trpC,bar, and hygB. Preferred for use in an Aspergillus cell are the amdS andpyrG markers of Aspergillus nidulans or Aspergillus oryzae and the barmarker of Streptomyces hygroscopicus. Furthermore, selection may beaccomplished by co-transformation, e.g., as described in WO 91/17243where the selectable marker is contained in a separate vector.

The vectors of the invention preferably also contain a signal peptidecoding region, which codes for an amino acid sequence linked to theamino terminus of the heme biosynthetic enzyme, permitting thelocalization of the 5-aminolevulinic acid synthase to a particularcellular compartment. The signal peptide coding region may be native tothe first nucleic acid sequence encoding the 5-aminolevulinic acidsynthase or may be obtained from foreign sources. The 5' end of thecoding sequence of the first nucleic acid sequence may inherentlycontain a signal peptide coding region naturally linked in translationreading frame with the segment of the coding region which encodes thelocalized 5-aminolevulinic acid synthase. Alternatively, the 5' end ofthe coding sequence may contain nucleic acids encoding a signal peptidecoding region which is foreign to that portion of the coding sequencewhich encodes the localized heme biosynthetic enzyme. The signal peptidecoding region may be obtained from a Neurospora crassa ATPase gene(Viebrock et al., 1982, EMBO Journal 1:565-571) or from a Saccharomycescerevisiae cytochrome c peroxidase gene (Kaput et al., 1982, Journal ofBiological Chemistry 257:15054-15058). However, any signal peptidecoding region capable of permitting localization of the 5-aminolevulinicacid synthase in a filamentous fungal host of choice may be used in thepresent invention.

To avoid the necessity of disrupting the cell to obtain the expressed5-aminolevulinic acid synthase, and to minimize the amount of possibledegradation of the expressed 5-aminolevulinic acid synthase within thecell, it is preferred that expression of the 5-aminolevulinic acidsynthase gene gives rise to a product secreted outside the cell. To thisend, the 5-aminolevulinic acid synthases of the present invention maythus comprise a preregion permitting secretion of the expressed proteininto the culture medium. If desirable, this preregion may be native tothe 5-aminolevulinic acid synthase of the invention or substituted witha different preregion or signal sequence, conveniently accomplished bysubstitution of the DNA sequences encoding the respective preregions.For example, the preregion may be obtained from a glucoamylase or anamylase gene from an Aspergillus species, an amylase gene from aBacillus species, a lipase or proteinase gene from Rhizomucor miehei,the gene for the α-factor from Saccharomyces cerevisiae or the calfpreprochymosin gene. Particularly preferred is the preregion forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, themaltogenic amylase from Bacillus NCIB 11837, Bacillus stearothermophilusα-amylase, or Bacillus licheniformis subtilisin. An effective signalsequence for fungal hosts is the Aspergillus oryzae TAKA amylase signal,the Rhizomucor miehei aspartic proteinase signal, or the Rhizomucormiehei lipase signal.

The procedures used to ligate the nucleic acid construct of theinvention, the promoter, terminator and other elements, and to insertthem into suitable vectors containing the information necessary forreplication, are well known to persons of ordinary skill in the art(cf., for instance, Sambrook et al., supra).

The present invention also relates to host cells comprising a nucleicacid construct or an expression vector of the invention which areadvantageously used in the recombinant production of the5-aminolevulinic acid synthases of the invention. The cell may betransformed with the nucleic acid construct of the invention,conveniently by integrating the construct into the host chromosome. Thisintegration is generally considered to be an advantage as the sequenceis more likely to be stably maintained in the cell. Integration of theconstruct into the host chromosome may be performed according toconventional methods, e.g., by homologous or non-homologousrecombination. Alternatively, the cell may be transformed with anexpression vector as described below in connection with the differenttypes of host cells.

The choice of host cells and vectors will to a large extent depend uponthe 5-aminolevulinic acid synthase and its source. The host cell may beselected from prokaryotic cells, such as bacterial cells. Examples ofsuitable bacteria are gram-positive bacteria such as Bacillus subtilis,Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium, Bacillus thuringiensis, or Streptomyces lividans orStreptomyces murinus, or gram-negative bacteria such as E. coli. Thetransformation of the bacteria may, for instance, be effected byprotoplast transformation or by using competent cells in a manner knownper se.

The host cell is preferably a eukaryote, such as a mammalian cell, aninsect cell, a plant cell or preferably a fungal cell, including yeastand filamentous fungi. For example, useful mammalian cells include CHOor COS cells. A yeast host cell may be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g., Saccharomyces cerevisiae.Useful filamentous fungi may be selected from a species of Aspergillus,e.g., Aspergillus oryzae or Aspergillus niger. Alternatively, a strainof a Fusarium species, e.g., Fusarium oxysporum or Fusarium graminearum,can be used as a host cell. Fungal cells may be transformed by a processinvolving protoplast formation, transformation of the protoplasts, andregeneration of the cell wall in a manner known per se. A suitableprocedure for transformation of Aspergillus host cells is described inEP 238 023. A suitable method of transforming Fusarium species isdescribed by Malardier et al, 1989, Gene 78:147-156 or in copending U.S.Ser. No. 08/269,449.

In a particularly preferred embodiment, the expression of the5-aminolevulinic acid synthase gene is achieved in a fungal host cell,such as Aspergillus. The 5-aminolevulinic acid synthase gene is ligatedinto a plasmid preferably containing the Aspergillus oryzae TAKA amylasepromoter or the Aspergillus niger neutral amylase NA2 promoter and amdSor pyrG as the selectable marker. Alternatively, the selectable markermay be on a separate plasmid and used in co-transformation. The plasmid(or plasmids) is used to transform an Aspergillus species host cell,such as Aspergillus oryzae or Aspergillus niger in accordance withmethods described in Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences U.S.A. 81:1470-1474.

The present invention also relates to methods for producing a5-aminolevulinic acid synthase of the present invention comprising (a)cultivating an Aspergillus oryzae strain in a nutrient medium to producethe 5-aminolevulinic acid synthase, and (b) recovering the5-aminolevulinic acid synthase.

The present invention also relates to methods for recombinantlyproducing a 5-aminolevulinic acid synthase of the present inventioncomprising (a) fermenting a host cell comprising a nucleic acidconstruct comprising a nucleic acid sequence encoding the5-aminolevulinic acid synthase under conditions conducive to theproduction of the enzyme, and (b) recovering the 5-aminolevulinic acidsynthase. If the expression system secretes the 5-aminolevulinic acidsynthase into the fermentation medium, the enzyme can be recovereddirectly from the medium. If the recombinant 5-aminolevulinic acidsynthase is not secreted, it is recovered from cell lysates.

Any method of cultivation of a cell known in the art may be used whichresults in the expression or isolation of a 5-aminolevulinic acidsynthetase of the present invention. For example, cultivation may beunderstood as comprising shake flask cultivation, small- or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the 5-aminolevulinic acidsynthase to be expressed or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts using procedures known in the art (see, e.g., Bennett,J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, California, 1991). Suitable media are available from commercialsuppliers or may be prepared according to published compositions (e.g.,in catalogues of the American Type Culture Collection).

The 5-aminolevulinic acid synthases produced by the methods describedabove may be recovered from the fermentation medium by conventionalprocedures including, but not limited to, centrifugation, filtration,spray-drying, evaporation, or precipitation. The recovered protein maythen be further purified by a variety of chromatographic procedures,e.g., ion exchange chromatography, gel filtration chromatography,affinity chromatography, or the like.

The present invention is also directed to methods of using the5-amininolevulinic acid synthases.

The 5-aminolevulinic acid synthases of the present invention may be usedto convert glycine and succinyl-CoA to 5-aminolevulinic acid which isuseful as a herbicide.

The 5-aminolevulinic acid synthases of the present invention may be alsoused to increase the yield of a hemoprotein produced by a host cell,where 5-aminolevulinic acid synthase is a rate-limiting step in theproduction of heme in the host cell, by overexpressing the nucleic acidsequence encoding the 5-aminolevulinic acid synthase in the host cell.The method comprises:

(a) introducing into the host cell, which is capable of producing thehemoprotein, one or more copies of the nucleic acid sequence encodingthe 5-aminolevulinic acid synthase, wherein the nucleic acid sequence isoperably linked to regulatory regions capable of directing theexpression of the 5-aminolevulinic acid synthase;

(b) cultivating the cell in a nutrient medium suitable for production ofthe hemoprotein and the 5-aminolevulinic acid synthase; and

(c) recovering the hemoprotein from the nutrient medium of the cell.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES Example 1 Aspergillus oryzae Strain A1560 Genomic DNAExtraction

Aspergillus oryzae strain A1560 (IFO 4177) was grown in 25 ml of 0.5%yeast extract-2% glucose (YEG) medium for 24 hours at 32° C. and 250rpm. Mycelia were then collected by filtration through Miracloth(Calbiochem, La Jolla, Calif.) and washed once with 25 ml of 10 mMTris-1 mM EDTA (TE) buffer. Excess buffer was drained from the myceliawhich were subsequently frozen in liquid nitrogen. The frozen myceliawere ground to a fine powder in an electric coffee grinder, and thepowder was added to 20 ml of TE buffer and 5 ml of 20% w/v sodiumdodecylsulfate (SDS) in a disposable plastic centrifuge tube. Themixture was gently inverted several times to insure mixing, andextracted twice with an equal volume of phenol:chloroform:isoamylalcohol (25:24:1 v/v/v). Sodium acetate (3 M solution) was added to afmal concentration of 0.3 M followed by addition of 2.5 volumes of icecold ethanol to precipitate the nucleic acids. The nucleic acids werethen pelleted by centrifuging the tube at 15,000×g for 30 minutes. Thepellet was allowed to air dry for 30 minutes before resuspension in 0.5ml of TE buffer. DNase-free ribonuclease A was added to a concentrationof 100 μg/ml and the mixture was incubated at 37uC. for 30 minutes.Proteinase K was then added at a concentration of 200 μg/ml and themixture was incubated an additional hour at 37° C. Finally, the mixturewas extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1v/v/v) before precipitating the DNA with sodium acetate and ethanol asdescribed earlier. The DNA pellet was dried under vacuum, resuspended inTE buffer, and stored at 4° C. until further use.

Example 2 Construction of Plasmid pSE04

Genomic DNA was obtained from Aspergillus nidulans strain A26 (FungalGenetics Stock Center, Kansas City, Kans.) using the same proceduredescribed in Example 1. Plasmid pSE04 was constructed by ligation of PCRfragments from an amplification reaction containing Aspergillus nidulansA26 genomic DNA. The amplification reaction contained the followingcomponents: 50 ng of Aspergillus nidulans A26 genomic DNA, 100 μM eachof dATP, dCTP, dGTP, and dTTP (Boehringer Mannheim, Indianapolis, Ind.),50 pmoles of primers ALAS3d 5'-TTTATGATGGAGGCCCTTCTCCAGCAGTCTC-3' (SEQID NO:3) and ALAS4e 5'-CTATGCATTTAAGCAGCAGCCGCGACTGG-3' (SEQ ID NO:4), 2units of Taq DNA polymerase (Perkin-Elmer Corp., Branchburg, N.J.), and1X Taq DNA polymerase buffer (Perkin-Elmer Corp., Branchburg, N.J.). Thereaction was incubated in a Perkin-Elmer Thermal Cycler (Perkin-ElmerCorp., Branchburg, N.J.) programmed for 30 cycles each at 95° C. for 1minute, 55° C. for 1 minute, and 72° C. for 90 seconds. The 2 kb PCRproduct was isolated by excision after electrophoresis using a 1.1% lowmelting temperature agarose gel (FMC, Rockland, Me.) with 40 mMTris-acetate-1 mM disodium EDTA (TAE) buffer, and subcloned into thepCRII vector (Invitrogen, San Diego, Calif.) according to themanufacturer's instructions to produce pSE04 (FIG. 1).

Example 3 Aspergillus oryzae Strain A1560 DNA Libraries andIdentification of ALA Synthase (hemA) Clones

Aspergillus oryzae strain A1560 genomic DNA libraries were constructedusing the bacteriophage cloning vector λZipLox (Life Technologies,Gaithersburg, Md.) according to the manufacturer's instructions using E.coli Y1090ZL cells as a host for plating and purification of recombinantbacteriophage and E. coli DH10Bzip for excision of individual pZLl-hemAclones. Total cellular DNA prepared as described in Example 1 waspartially digested with Tsp5009I and size-fractionated on a 1% agarosegel with 50 mM Tris-50 mM borate-1 mM disodium EDTA (TBE) buffer. DNAfragments migrating in the size range 4-7 kb were excised and elutedfrom the gel using Prep-a-Gene reagents (BioRad Laboratories, Hercules,Calif.). The eluted DNA fragments were ligated with EcoRI-cleaved anddephosphorylated λZipLox vector arms, and the ligation mixtures werepackaged using commercial packaging extracts (Stratagene, La Jolla,Calif.). The packaged DNA libraries were plated and amplified in E. coliY1090ZL cells. The unamplified genomic library contained 1×10⁶ pfu/ml.

Bacteriophage DNA from 7×10⁴ plaques was transferred to duplicatecircular Nytran Plus membranes (Schleicher & Schuell, Keene, N.H.) andprobed with a digoxigenin (DIG)-labeled probe which was prepared by PCRamplification of Aspergillus nidulans hemA genomic DNA from plasmidpSE04 described in Example 2. The amplification reaction contained thefollowing components: 1X DIG probe synthesis mix (Boehringer Mannheim,Indianapolis, Ind.), 100 μM each of dATP, dCTP, dGTP, and dTTP, 50pmoles of primer ALAS3d and primer ALAS4e described in Example 2, 2units of Taq DNA polymerase, and 1X Taq DNA polymerase buffer. Thereaction was incubated in a Perkin-Elmer Thermal Cycler programmed for30 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C.for 2 minutes. Denatured probe was added to the hybridization buffer ata concentration of 2 ng/ml and incubated overnight with prehybridizedmembranes. Prehybridization and hybridization was conducted at 42° C. in5 X SSC, 0.1% sarkosyl, 0.02% SDS, 1% Genius blocking agent (BoehringerMannheim, Indianapolis, Ind.), and 30% formamide. Membranes were washedtwice in 5 X SSC-0.1% SDS followed by two washes in 2 X SSC-0.1% SDS.Each wash was performed for 15 minutes at room temperature. The washedmembrane was exposed to Kodak X-OMAT AR film for approximately 2 hoursat room temperature followed by development using a Konica QX-70automatic film processor according to the manufacturer's instructions.Primary plaques were purified and screened a second time. Five cloneswere identified and excised into pZL derivatives according to themanufacturer's instructions (Bethesda Research Laboratories, Inc.,Gaithersburg, Md.). The pZL derivatives were designated E. coliDH5αpSE11, pSE13, pSE15, pSE17, and pSE20. These clones were found tooverlap and span a 4.2 kb region for which the restriction map is shownin FIG. 2.

Example 4 Southern Hybridization of Aspergillus oryzae Strain A1560Genomic DNA with a 5-aminolevulinic Acid Synthase (hemA) Probe

Aspergillus oryzae strain A1560 genomic DNA (10 μg) prepared asdescribed in Example 1 was restriction digested with either BamHI orEcoRI. The fragments were separated by electrophoresis on a 1%agarose-TBE gel. DNA was transferred to a Nytran Plus membrane in 0.4 NNaOH using a TurboBlot apparatus (Schleicher & Schuell, Keene, N.H.)according to the manufacturer's instructions. The membrane wasprehybridized for 2 hours at 42° C. in 5 X SSC, 0.1% sarkosyl, 0.02%SDS, 1% Genius blocking agent (Boehringer Mannheim, Indianapolis, Ind.),and 50% formamide in a Hybaid oven (Labnet, Woodbridge, N.J.).Hybridization was accomplished with a DIG-labeled hemA probe generatedby PCR amplification as described in Example 3, except the hemA clonepSE17 was used as a template with primer hemA5'5'-TCATTTAAATGATGGAGTCTCTTCTCC-3' (SEQ ID NO:5) and primer hemA3'5'-TCTTAATTAATCAGCTCACATGCGGG-3' (SEQ ID NO:6). DIG-labeled hemA probe(1 ng probe/ml of solution) was added to fresh hybridization buffer andincubated with the membrane overnight at 42° C. Subsequently, themembrane was washed twice for 15 minutes each at room temperature in 5 XSSC-0.1% SDS followed by two washes under the same conditions in 2 XSSC-0.1% SDS. The washed membrane was exposed to Kodak X-OMAT AR filmfor approximately 2 hours at room temperature followed by developmentusing a Konica QX-70 automatic film processor according to themanufacturer's instructions.

Southern blot hybridization of Aspergillus oryzae genomic DNA with theAspergillus oryzae hemA probe showed the presence of hybridizationsignals consistent with a single gene copy number. A 1.7 kb bandobserved in the BamHI lane was predicted from the restriction map (FIG.2).

Example 5 Characterization of Aspergillus oryzae A1560 5-aminolevulinicAcid Synthase (hemA) Gene

E. coli DH5αpSE17 described in Example 3 was subjected to DNA sequencingaccording to the following procedure. DNA sequencing was performed withan Applied Biosystems Model 373A Automated DNA Sequencer (AppliedBiosystems, Inc., Foster City, Calif.) on both strands using the primerwalking technique with dye-terminator chemistry (Giesecke et al., 1992,Journal of Virol. Methods 38:47-60) using the M13 reverse (-48) and M13forward (-20) primers (New England Biolabs, Beverly, Mass.) and primersunique to the DNA being sequenced.

The nucleotide sequence of the cloned gene revealed an open readingframe of 1911 nucleotides as shown in FIG. 3 (SEQ ID NO:1). The codingsequence does not contain any introns which was confirmed by cDNAcloning and sequence analysis which is in contrast to the Aspergillusnidulans hemA gene which contains one intron at its 5' end (Bradshaw etal., 1993, Current Genetics 23:501-507). The 5' untranslated sequencecontains several pyrimidine-rich and AT-rich regions as in other fungalgenes (Gurr et al., 1987, In Kinghorn, J. R. (ed.), Gene Structure inEukaryotic Microbes, pp. 93-139, IRL Press, Oxford), a CCAAT sequence atposition -249, and a putative TATA box located at position -35. TheCCAAT sequence is a consensus binding site for transcriptionalregulators which modulate transcription in response to oxygen, such asthe Hap2/3/4 transcriptional regulatory complex in yeast and humans(Olesen and Guarente, 1990, Molecular and Cellular Biology12:2302-2314). This regulatory complex is also conserved in mammals, anda CCAAT-binding activity has been identified in Aspergillus nidulans(Davis et al., 1993, Genetica 90:133-145). The importance of thissequence in the Aspergillus oryzae hemA gene is not known and, due tolimited sequence information, has not been confirmed in the Aspergillusnidulans hemA 5' region (Bradshaw et al., 1993, supra). Transcriptionalregulation of the Aspergillus oryzae hemA gene in response to oxygen isnot currently known, but the Aspergillus nidulans hemA gene does notappear to be transcriptionally regulated even under conditions of oxygenlimitation (Bradshaw et al., 1993, supra). Interestingly, the yeast HEMlgene is also constitutively expressed, but its expression is controlledby a balance between positive and negative regulatory sites (Keng andGuarente, 1987, Proceedings of the National Academy of Sciences U.S.A.84:9113-9117). An (AC)₃₅ repeat motif occurs in the 3' untranslatedregion. Similar repeats have also been observed in subtelomeric, intron,and promoter regions of mammalian and yeast genes and have no knownfunction, although they have been implicated in gene amplificationevents (Passananti et al., 1987, EMBO Journal 6:1697-1703).

The deduced amino acid sequence of the Aspergillus oryzae strain A1560gene product is shown in FIG. 3 (SEQ ID NO:2). The nucleotide sequenceencodes a predicted protein of 636 amino acids with a molecular weightof 68 kDa. Since this enzyme is located in the mitochondria, theN-terminus is predicted to contain a mitochondrial leader sequence. Infact, the first 35 amino acids are rich in serine, threonine, lysine,and arginine residues consistent with a function as a mitochondrialleader. A potential heme regulatory motif (HRM) occurs in the presumedmitochondrial leader sequences of both the Aspergillus nidulans andAspergillus oryzae hemA sequences (FIG. 4). HRMs localized to leadersequences are believed to prevent import of 5-aminolevulinic acidsynthase proteins into the mitochondria in mouse via direct interactionswith heme (Lathrop and Timko, 1993, Science 259:522-525; Zhang andGuarente, 1995, EMBO Journal 14:313-320). A second potential HRM alsooccurs in the beginning of the putative mature protein sequence. It isprobable that the HRMs play a role in the regulation of 5-aminolevulinicacid synthase activity. Interestingly, the Saccharomyces cerevisiae5-aminolevulinic acid synthase protein sequence does not contain anyputative HRMs and does not appear to be a key regulatory step in yeastheme biosynthesis (Labbe-Bois and Labbe, In Daley, Harry A., ed.,Biosynthesis of Heme and Chlorophylls, 1990, McGraw Hill Publishers, NewYork, pp 235-285).

Overall, the deduced amino acid sequence as shown in FIG. 5 shares 81%identity with the Aspergillus nidulans hemA gene (SEQ ID NO:16), 57%identity with the Saccharomyces cerevisiae HEMl gene (SEQ ID NO:17;Urban-Grimal, 1986, European Journal of Biochemistry 156:511-519), and51% identity with the human erythroid heml (ALAS2) gene (SEQ ID NO:18;Bishop, 1990, Nucleic Acids Research 18:7187-7188) which were determinedusing the Applied Biosystems GeneAssist program (blosum62.mat matrix).However, the highest degree of conservation occurs in the C-terminaltwo-thirds of the protein which contains the catalytic domain.Furthermore, the lysine and glycine-loop, important for catalyticactivity and pyridoxal phosphate co-factor binding in other5-aminolevulinic acid synthase enzymes (Ferreira et al., 1995, Journalof Bioenergetics and Biomembranes 27:151-159; Ferreira, 1995, ProteinScience 4:1001-1006) are also highly conserved.

Example 6 Construction of Plasmid pSE31

Plasmid pSE31 was constructed by directional cloning of PCR-amplifiedAspergillus oryzae hemA DNA into pBANe6 (FIG. 6). The PCR amplificationreaction was performed using DNA from hemA clone E. coli DH5α pSE17described in Example 3 where the reaction contained the followingcomponents: 50 ng of pSE17, 2 units of Vent DNA polymerase (New EnglandBiolabs, Beverly, Mass.), 1X Vent DNA polymerase buffer (New EnglandBiolabs, Beverly, Mass.), 400 μM each of dATP, dCTP, dGTP, and dTTP(Boehringer Mannheim, Indianapolis, Ind.), and 50 pmoles of primerhemA5'5'-TCATTTAAATGATGGAGTCTCTTCTCC-3' (SEQ ID NO:5) and primerhemA3'5'-TCTTAATTAATCAGCTCACATGCGGG-3' (SEQ ID NO:6). The reaction wasincubated in a Perkin-Elmer Thermal Cycler programmed for 30 cycles eachat 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 90 seconds.Primer hemA5' contains a SwaI site (underlined) and primer hemA3'contains a PacI site (underlined) which were used for cloning intopBANe6 digested with Swal and PacI to produce pSE31 (FIG. 7).

Example 7 Construction of Aspergillus oryzae Strain JRoC50.3.18A

Aspergillus oryzae strain JRoC50.3.18A containing plasmid pJROC50 wasconstructed as follows. Coprinus cinereus IFO 8371 peroxidase cDNAfragments were prepared by PCR using specific oligonucleotide primersshown below (Saiki et al., 1988, Science 239:487-491) constructed on thebasis of the amino acid sequence of the Coprinus macrorhizus peroxidase(Baunsgaard et al., 1993, European Journal of Biochemistry 213:605-611):

1. 5'-GCGCGAATTCGTNGGNATNGGNATNAA(CT)CA(CT)GG-3' (SEQ ID NO:7)

2. 3'-TACAGNTT(GA)AC(GA)GGNGGCCTAGGCG-5' (SEQ ID NO:8)

3. 5'-GCGAATTCACNCCNCA(GA)GTNTT(CT)GA(CT)AC-3' (SEQ ID NO:9)

4. 3'-GGNAA(GA)GGNCCNCT(CT)AA(GA)CCTAGGCG-5' (SEQ ID NO:10)

5. 5'-GCGCGAATTCTGGCA(GA)TCNAC-3' (SEQ ID NO:11)

6. 5'-GCGCGAATTCTGGCA(GA)AGNATG-3' (SEQ ID NO:12)

7. 3'-CGNTACCGNTT(CT)TACAGCCTAGG-5' (SEQ ID NO:13)

PCR was performed using the Gene Amp Kit and apparatus (Perkin ElmerCetus, Norwalk, Conn.) in accordance with the manufacturer'sinstructions with the exception that the reaction was conducted at 28°C. for the first 3 cycles in order to obtain better hybridization to thefirst strand cDNA (prepared from mRNA obtained from Coprinus cinereusstrain IFO 8371) and subsequently at 65° C. for 30 cycles of PCR.

The primers were combined as follows: 1 with 2; 3 with 4; 5 with 7; 6with 7; 1 with 4; and 3 with 7. The PCR fragments were extended with anEcoRI site at the 5'-end and a BamHI site at the 3'-end. The reactionswere analyzed on a 1% agarose-TBE gel where bands of the expected sizewere found in all the reactions. To verify that the bands correspondedto peroxidase-specific sequences, the gel was subjected to Southernblotting and hybridized to an oligonucleotide probe with the followingsequence which is positioned between primers 3 and 4:

    5'-GT(CT)TC(GA)AT(GA)TAGAA(CT)TG-3'                        (SEQ ID NO:14)

The probe was found to hybridize to bands of approximately 130 bp, 420bp, 540 bp, and 240 bp, thus confirming that the DNA bands observedcorresponded to peroxidase sequences.

DNA from the various PCR reactions was digested with EcoRI and BamHI andcloned into the plasmid pUC19 (New England BioLabs, Beverly, Mass.).Colonies containing the correct PCR fragments were identified byhybridization using the oligonucleotide probe (SEQ ID NO:14) describedabove. DNA from positive colonies was analyzed by restriction mappingand partial DNA sequence analysis as described by Sanger et al. (1977,Proceedings of the National Academy of Sciences U.S.A. 74:5463-5467). A430 bp fragment from one of the clones, obtained by using primers 1 and4, was used to screen a Coprinus cinereus cDNA library as describedbelow.

Total RNA was extracted from homogenized Coprinus cinereus strain IFO8371 mycelia, collected at the time of maximum peroxidase activityaccording to the methods described by Boel et al. (1984, EMBO Journal3:1097-1102) and Chirgwin et al. (1979, Biochemistry 18:5294-5299).Poly(A)-containing RNA was obtained by two cycles of affinitychromatography on oligo(dT)-cellulose as described by Aviv and Leder(1972, Proceedings of the National Academy of Sciences U.S.A.69:1408-1412). cDNA was synthesized by means of a cDNA Synthesis Kit(Invitrogen, San Diego, Calif.) according to the manufacturer'sinstructions. Approximately 50,000 E. coli recombinants from theCoprinus cinereus cDNA library were transferred to Whatman 540 paperfilters. The colonies were lysed and immobilized as described by Gergeret al. (1979, Nucleic Acids Research 7:2115-2135). The filters werehybridized with the ³² P-labelled 430 bp peroxidase-specific probe in0.2 X SSC-0.1% SDS. Hybridization and washing of the filters wasconducted at 65° C. followed by autoradiography for 24 hours with anintensifier screen. After autoradiography, the filters were washed atincreasing temperatures followed by autoradiography for 24 hours with anintensifier screen. In this way, more than 50 positive clones wereidentified. Miniprep plasmid DNA was isolated from hybridizing coloniesby standard procedures (Birnboim and Doly, 1979, Nucleic Acids Research7:1513-1523), and the DNA sequences of the cDNA inserts were determinedby the Sanger dideoxy procedure (Sanger et al., 1977, Proceedings of theNational Academy of Sciences U.S.A. 74:5463-5467). One of the colonieswas selected and the vector was designated pCiP. The peroxidase cDNAfragment was excised from the vector by cleavage with BamHI/XhoI and waspurified by agarose gel electrophoresis, electroeluted and made readyfor ligation reactions. The cDNA fragment was ligated to BamHI/XhoIdigested pHD414 to generate pJVi9 wherein the cDNA was undertranscriptional control of the TAKA promoter from Aspergillus oryzae andthe AMG™ (Novo Nordisk A/S, Bagsv.ae butted.rd, Denmark) terminator fromAspergillus niger as shown in FIG. 8.

The cDNA encoding the Coprinus cinereus peroxidase was excised fromplasmid pJVi9 as a BamHI-XhoI fragment and cloned into plasmid pJeRS6(FIG. 9) to produce plasmid pJRoC50 (FIG. 10) which contains pyrG as aselectable marker, the TAKA promoter, and the amdS terminator.

Transformants of Aspergillus oryzae strain HowB425 were made using 5 μgof purified plasmid pJRoC50 as described below with the followingchanges. The agar overlay was omitted and the protoplasts were plateddirectly on Minimal Medium plates. The transformation was conducted withprotoplasts at a concentration of 2×10⁷ protoplasts per ml. One hundredμl of protoplasts were placed on ice with 5 μg DNA for 30 minutes. Oneml of SPTC (40% PEG 4000, 0.8 M sorbitol, 0.05 M Tris pH 8.0, 0.05 MCaCl₂) was added and the protoplasts were incubated at 34° C. for 20minutes. The transformation was plated directly onto plates containingMinimal medium. The Minimal medium (pH 6.5) was composed of 6 g ofNaNO₃, 0.52 g of KCl, 1.52 g of KH₂ PO₄, 1 ml of trace metals, 1 g ofglucose, 500 mg of MgSO₄ -7H₂ O, 342.3 g of sucrose, and 20 g of Nobleagar per liter. The trace metals solution (1000X) was composed of 22 gof ZnSO₄ -7H₂ O, 11 g of H₃ BO₃, 5 g of MnCl₂ -4H₂ O, 5 g of FeSO₄ -7H₂O, 1.6 g of CoCl₂ -5H₂ O, 1.6 g of (NH₄)₆ Mo₇ O₂₄, and 50 g of Na₄ EDTAper liter. Plates were incubated 5-7 days at 34° C. Transformants weretransferred to plates of the same medium and incubated 3-5 days at 37°C.

Sixty-six transformants were assayed for peroxidase activity using thefollowing enzyme assay: 180 μl of substrate buffer {20 ml of 0.1 Mpotassium phosphate-0.01% Tween-80 pH 7.0, 250 μl of2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) solution (22mg/ml), and 2 μl of 30% hydrogen peroxide} were added to 20 μl ofculture supernatant which was diluted 1:900, quickly followed bymeasurement of the absorbance at 405 nm at 25° C. using a MolecularDevices Thermomax Microplate Reader (Molecular Devices, Sunnyvale,Calif.). Measurements were recorded every 10 seconds over a 2 minuteperiod with mixing and V_(max) values were calculated using the SOFTmaxprogram (Molecular Devices, Sunnyvale, Calif.). The peroxidase units(POXU) per ml were estimated using a standard curve constructed with aknown amount of Cinereus coprinus peroxidase as a standard. A POXU wasdefined as the amount of enzyme that catalyzes the conversion of 1.0μmole per minute of 0.88 mM H₂ O₂, 1.67 mM ABTS, 0.1 M phosphate pH 7.0at 30° C. The four transformants expressing the highest levels werespore purified by streaking spores and picking isolated colonies usingthe same plates under the same conditions described above.

Final evaluations were performed in shake flasks where approximately5×10⁶ spores of each transformant were inoculated into 25 ml of MY25medium containing 1% yeast extract, 2.5% maltose, 0.2% urea, and 1X MYsalts pH 6.5. 1X MY salts was composed of 2 g of MgSO₄ -7H₂ O, 2 g of K₂PO₄, 10 g of KH₂ PO₄, 2 g of citric acid, 0.5 ml of trace metalssolution and 1 ml of 10% CaCl₂ -2H₂ O per liter. The trace metalssolution was composed of 13.9 g of FeSO₄ -7H₂ O, 8.5 g of MnSO₄ -H₂ O,14.28 g of ZnSO₄ -7H₂ O, 1.63 g of CuSO₄, 0.24 g of NiCl₂ -6H₂ O, and3.0 g of citric acid per liter. Hemin was added to a final concentrationof 0.01 mg/ml from a fresh 10 mg/ml stock prepared in 50 mM NaOH. Theshake flasks were incubated at 34° C. and 200 rpm for 7 to 8 days. Thebest peroxidase producer was designated JRoC50.3.18A.

Example 8 Transformation of Aspergillus oryzae JRoC50.3.18A with pSE31

Aspergillus oryzae strain JRoC50.3.18A was transformed with pSE31 inorder to determine whether overexpression of the hemA gene increasedperoxidase production.

The transformation was conducted with protoplasts at a concentration of2×10⁷ protoplasts per ml. One hundred μl of protoplasts were incubatedat 34° C. with 10 μg DNA and 200 μl of 60% PEG 4000-10 mM HEPES-10 mMCaCl₂ solution for 30 minutes. Three ml of SPTC (40% PEG 4000, 0.8 Msorbitol, 0.05 M Tris pH 8.0, 0.05 M CaCl₂) were added and theprotoplasts were plated directly onto COVE transformation plates (perliter: 0.52 g of KCl, 0.52 g of MgSO₄ -7H₂ O, 1.52 g of KH₂ PO₄, 1 ml oftrace metals solution as described in Example 7, 342.3 g of sucrose, 25g of Noble agar, 10 ml of 1 M acetamide, and 10 ml of 3 M CsCl) for amdStransformations. Plates were incubated 5-7 days at 34° C. Transformantswere transferred to plates of the same medium and incubated 3-5 days at34° C. The transformants were then purified by streaking spores andpicking isolated colonies using the same plates under the sameconditions.

Example 9 Peroxidase Production by hemA Transformants

The transformants from Example 8 were inoculated into individual wellsat approximately 1×10⁵ spores per well of a 24-well microtiter platecontaining 1 ml of quarter strength MY25 medium composed of 0.25% yeastextract, 0.63% maltose, and 0.05% urea pH 6.5, and 1X MY salts (seeExample 7). The microtiter plates were incubated at 34° C. and 100 rpmin a humidity chamber for 5 days.

Peroxidase production levels were determined using the enzyme assaydescribed in Example 7. The results of the microtiter plate testsdemonstrate that the average POXU/ml of hemA transformants was 1.4-foldgreater than the average of the vector only transformants, with the besthemA transformant showing a 1.6-fold increase in peroxidase production.

A minority (39%) of the hemA transformants show peroxidase levelssimilar to the majority of the vector only controls. PCR amplificationusing 50 ng of genomic DNA isolated as described in Example 1 from eachtransformant was performed as described in Example 2 except the primershemA3' (see Example 4) and primer 5'-TCTCTTCCTTCCTGAATCCTC-3' (SEQ IDNO:15) were used. This analysis showed that the hemA transformantscontain the expression cassette.

Eleven of the best hemA transformants obtained above were cultivated inshake flasks to better evaluate the effects on peroxidase production.For shake flask evaluations, approximately 5×10⁶ spores of eachtransformant were inoculated into 25 ml of MY25 medium containing 1%yeast extract, 2.5% maltose, 0.2% urea, and 1X MY salts pH 6.5 (seeExample 7). The shake flasks were incubated at 34° C. and 200 rpm for 7to 8 days. Peroxidase assays were performed as described above.

The results demonstrated that five transformants, SE01-15, SE01-20,SE01-26, SE01-28 and SE01-32, produced peroxidase levels which weregreater than the vector alone control strains, with three transformantsexpressing peroxidase at a level 1.9-fold greater than the averagecontrol peroxidase levels. The remaining six hemA transformants showedperoxidase levels which were comparable to control levels.

Transformant SE01-28 and a control strain SE05-18 (pBANe6 vector alonetransformant) were grown in 2 liter fermentations using a standardfed-batch protocol which has high maltose syrup as carbon source. Thebatch and feed were supplemented with FeCl₃ to approximately 0.4 mM.Positive dissolved oxygen tension was maintained in both cultures withfeed added at a rate of approximately 2 grams saccharide per liter perhour from day three to day eight. This level was reached in a step-wisemanner over days two and three. Biomass in both cultures wereapproximately equal for the duration of the fermentation.

A 2-fold increase in peroxidase activity was observed with SE01-28 overthe control strain SE05-18. There was also a 2-fold increase in thepolypeptide level for SE01-28 relative to the control strain SE05-18.

The overall results demonstrated that overexpression of the hemA generesulted in a 2-fold increase in peroxidase yield. The data indicatedfurther that hemA may represent a key regulatory point during hemebiosynthesis in filamentous fingi which upon genetic manipulation canimprove hemoprotein production in the absence of hemin supplementation.

DEPOSIT OF MICROORGANISMS

The following strain has been deposited according to the Budapest Treatyin the Agricultural Research Service Patent Culture Collection (NRRL),Northern Regional Research Laboratory, 1815 University Street, Peoria,Ill. 61604, U.S.A.

    ______________________________________    Strain         Accession Number                                Deposit Date    ______________________________________    E. coli DH5α (pSE17)                   NRRL B-21563 April 22, 1996    ______________________________________

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of eachdeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 18    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 4157 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    #ACAGGTCACG    60AAGCTAT GGATCGTGCT CACCGTCTCG GCCAGACAAG    #AGCTTTGCAG   120TTACTCG CGGCACCATT GAGGAGCGTA TTCGCAAGCG    #TGACTTCAAT   180AGCGTGT CGTCATCTCA GGTGGCGCAG CTGGTGGGGT    #TGATGAACAG   240AGAGCCG AACCAAGGAC ATCGCCATGT GGCTGGCAGA    #GTTTGGCGCT   300AGCAAAA GGAGAAGGAA GCGCTGGACC GAGGCGAAGT    #GGATGATATG   360AGAAGGC TGCTCAGAAG AGAAAGAGAG ATATCACGCT    #TCTGGAAATA   420TGTGAAT CTGATCAAAG CTCTTCGTTC CGGGGAGGCT    #CAAAGCCATC   480CAATCTA TAGGCGAAGG GAACTTTGAC GATGCCAGTG    #CCACGCCAGT   540CCTGTGT CGACTGCAGA GAATTTAGGC ACCCCATCCT    #CCAAAACTAC   600CGTGGAA GGGGGACAGG AAAGGGCACG TCTAAAAGAG    #GATTTAATCG   660CGTCTCA TTGATGGCGA CGGAGGCTTA GGGCCTAGTT    #ATTGCATTTC   720TAATGGA CACGGCTGGT TATGGTCATG GCGTTCAGAG    #TTTTCTTTTT   780ATCTTTC TTTCTTTCCT CTTAAACCCC TCTTTTTTGT    #ATATTCCTGA   840TGTGGGC AGCTTACGTT CTGCCTTGTA TTAACAGCAT    #CCGTGGTAAT   900AAGCGAT TTAAGAGTCA TTGAAGACGA AGGATGAAAC    #TGAAATTGAT   960CAAAGAG AAGGAGAAGA AAAAAATCAA GTGCGAGTTT    #GGTGAAATTG  1020TTGTATC CTGTACCTGT TCTTGGGCTG TGACGGGGGG    #CTGCTGGTCT  1080GGCTATT ATTACTATTG TTGTACTGTA CATCCGGATC    #TGATTTCATA  1140GCAATAT TCCCCGTCGC CAGGCCTCTT GGGTTATGAA    #ATTATGAAAA  1200ATCCGTA CGCACCGAGA GATTTCTTAG TATTACTTGT    #AGCCGAAAAG  1260TTAAGTC CGCCGGCCAA TCACGGCGGA GGATATGGTA    #AGCGCCCCCT  1320CCCGACT TACTCTTACT GGAAGTGGCT TAGTGCCCTC    #CGCGGGTGAC  1380TCAGCCA GATTGACTCT TATTTCTCTC TCCTCTTCGC    #CCTTTTGTGA  1440TCTCCCT CTCCCTCTTG ACAACATTTC ATCTTCGCTT    #CAGCAGTCCC  1500GCTATCC ATTGAAGCAT CACTCATGGA GTCTCTTCTC    #CTGGCAACCG  1560GTTCCTT AAGCGCACAT CTCCATCTTC TCTGCGTACG    #GTCATTGCCC  1620CACTAGT TCCGGTGGAG GCACTATGTC TAATCTCCAG    #GCCGGTACCA  1680CATGAGC AAGGCTCTGG CCGTGCAGAG CGCTCGCATG    #TGCCGTGCTC  1740ATGTGCT GCCGGCATCA CCGGTCTCGG CAACAAGCAT    #AGCGCAGAGA  1800AACCCTG CACTCCACCT CCGGTAACGG CGCCAATGTG    #CCTGCCAATG  1860CCAGCGA GATCCCGCCG GTTTCTCGAA GATCAAGACC    #AAGCCTTTCA  1920CGCTACG TCTGGCCCTC GTCCAGAGGC TCCCGTGGCG    #TCGTATCGCT  1980CTACAAC ACCGAATTGG AAAAGAAACA CAAGGACAAG    #ACATCTGCCG  2040CAATCGT CTCGCTCAGG AGTTTCCCCG GGCTCACACC    #AACCCCGAGG  2100GGTCTGG TGCTCGAACG ATTATCTCGG CATGGGCCGC    #GGTACTCGCA  2160GCATAAG ACATTGGACA CCTACGGAGC CGGTGCGGGA    #AAATTGCACG  2220CAATCAA CATGCCGTGA GCCTGGAGAA CACCCTGGCC    #ACCCTCGCAA  2280ATTAGTC TTCAGCTCAT GCTTCGTGGC TAACGATGCC    #CATGCATCGA  2340GTTGCCC GACTGTGTTA TTCTGTCCGA TAGCCTGAAT    #AATGATCTGG  2400TCGCCAT TCAGGCGCCA AGAAAATGGT TTTCAAGCAT    #ATTGCATTCG  2460CAAGTTG GCAGCTCTAC CTCTTCATGT CCCCAAGATT    #GATCTTGCAG  2520CATGTGC GGATCTATTG CCCCAATTGA GAAGATCTGT    #TACGGACCTC  2580CATTACT TTCCTGGATG AAGTCCACGC TGTGGGAATG    #GATACGGTCA  2640GGCAGAG CACCTTGACT ATGACATCTA TGCTTCCCAA    #GGTACTCTGG  2700TAAGGGA ACCGTGATGG ACCGAATCGA TATTATCACC    #GTTGACACCA  2760ATGTGTC GGGGGCTACA TTGCTGGATC CGCTGCGATG    #ACCATGGCTG  2820CCCTGGC TTCATCTTCA CCACGTCCTT GCCGCCCGCC    #CTGCAGCAGT  2880TATCCAG TACCAGGCTC GTCACCAGGG CGACCGCGTC    #ATTCCCAACC  2940GGTCAAA GCAGCTTTCA AGGAGTTGGA TATTCCTGTA    #GCCTCGGACA  3000TCCGCTC CTGGTTGGGG ATGCCGAGGT TGCTAAGAAG    #GTGCCTCGGG  3060GCATGGA ATTTATGTAC AAGCCATCAA CTACCCAACC    #CGCGACCACC  3120TCGTATC ACGCCCACCC CGGGACATAT CAAGGAGCAC    #AGCGATTGGG  3180CCAAACA GTCTGGAACG AACTGGGCAT CAAACGCACC    #AACCAGCCGA  3240CTTCGTC GGCGTGGGTG TCGATGGCGC CGAGGCTGAG    #GCTGTGGAAC  3300GCAGCTG GGGCTGAAGG AAAACGAAGC CATTGAGGCT    #GCTGCTTCGT  3360GGCCCCC ATGCGGACCG CCACCCGTCC TGCCGCGGCT    #AATCGACGTG  3420TGTGGCT GCCTGAAGTG GCTGCCCGCA TGTGAGCTGA    #CACACACACA  3480ACACACA CACACACACA CACACACACA CACACACACA    #ACTCCTTTGT  3540ACACACT AACACACACT ATGTTATAAA TTCCACATCC    #CGTTACCATG  3600TAATTGG TATTTGGACT ATTAGTTAGA ACCAGTCAGT    #CATCTCCTCC  3660CTCGAAA TCTGACATGT TGTCTGCCCC CATGCCACTT    #AGTTAACTAA  3720TTCAAAT ACACTGCCCA GTAATTGTAG TCAATATAGC    #AAGATCACCT  3780CCTAATA ACAATAGAAG GGGCCATACA CGCAGTACCA    #TCGATTCCGA  3840ATCCGAA CCTCAGGCTA CATACATCAA GTCGCATTAA    #TGGGACACCT  3900CTGAAAA TAACTAAGAT CATGATCTAC GTTTGGTAAG    #TGCCAGGGCC  3960GGTATTG AATAAAGGCA TCATTCATAT AGTCACAAGA    #TGCTCTTCAG  4020GATAGCT ACTTCCAAAC ATAATTCAGA GGTATCATTC    #ATGTAATTGC  4080AAGATCA GTAGGAGCCA GTTTTGACCA TTAACTTGTA    #TTTTACTGAT  4140CCGAGAT CCATTCACTT TCTAAGGGTT AATTGATTCA    # 4157             TT    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 636 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -      (v) FRAGMENT TYPE: internal    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    #Met Cys Pro Phe Leu Lysln Gln Ser Arg Ala    #                 15    #Ala Thr Ala Thr Arg Proer Leu Arg Thr Leu    #             30    #Asn Leu Gln Val Ile Alaly Gly Thr Met Ser    #         45    #Ala Val Gln Ser Ala Arget Ser Lys Ala Leu    #     60    #Ala Ala Gly Ile Thr Glyrg Phe Thr Ser Cys    # 80    #Gly Lys Arg Thr Leu Hisys Arg Ala Pro Thr    #                 95    #Ala Glu Ile Tyr Lys Asnly Ala Asn Val Ser    #            110    #Ile Lys Thr Pro Ala Asnla Gly Phe Ser Lys    #        125    #Arg Pro Glu Ala Pro Valla Thr Ser Gly Pro    #    140    #Asn Thr Glu Leu Glu Lysyr Asn Ser Phe Tyr    #160    #Asn Asn Ile Asn Arg Leuer Tyr Arg Tyr Phe    #                175    #Ser Ala Glu Glu Arg Valrg Ala His Thr Thr    #            190    #Met Gly Arg Asn Pro Glusn Asp Tyr Leu Gly    #        205    #Thr Tyr Gly Ala Gly Alais Lys Thr Leu Asp    #    220    #Gln His Ala Val Ser Leule Ser Gly His Asn    #240    #Glu Ala Ala Leu Val Pheys Leu His Gly Lys    #                255    #Leu Ala Thr Leu Gly Serla Asn Asp Ala Thr    #            270    #Ser Leu Asn His Ala Seral Ile Leu Ser Asp    #        285    #Lys Lys Met Val Phe Lysrg His Ser Gly Ala    #    300    #Leu Ala Ala Leu Pro Leusp Leu Glu Ala Lys    #320    #Val Tyr Ser Met Cys Glyle Ala Phe Glu Ser    #                335    #Leu Ala Asp Lys Tyr Glylu Lys Ile Cys Asp    #            350    #Val Gly Met Tyr Gly Prosp Glu Val His Ala    #        365    #Tyr Asp Ile Tyr Ala Serla Glu His Leu Asp    #    380    #Gly Thr Val Met Asp Argro Arg Ser Thr Lys    #400    #Ala Tyr Gly Cys Val Glyly Thr Leu Gly Lys    #                415    #Asp Thr Ile Arg Ser Leuer Ala Ala Met Val    #            430    #Pro Pro Ala Thr Met Alahe Thr Thr Ser Leu    #        445    #Arg His Gln Gly Asp Argle Gln Tyr Gln Ala    #    460    #Lys Ala Ala Phe Lys Gluis Thr Arg Ala Val    #480    #His Ile Ile Pro Leu Leule Pro Asn Pro Ser    #                495    #Ser Asp Lys Leu Leu Glual Ala Lys Lys Ala    #            510    #Tyr Pro Thr Val Pro Argal Gln Ala Ile Asn    #        525    #Pro Gly His Ile Lys Glurg Ile Thr Pro Thr    #    540    #Thr Val Trp Asn Glu Leual Gln Ala Val Gln    #560    #Gln Gly Gly Phe Val Glyer Asp Trp Glu Ala    #                575    #Gln Pro Ile Trp Asn Aspla Glu Ala Glu Asn    #            590    #Ile Glu Ala Ala Val Gluys Glu Asn Glu Ala    #        605    #Ala Thr Arg Pro Ala Alala Pro Met Arg Thr    #    620    #Ala Alala Ala Ser Ser Ile Pro Val Gly Val    #635    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 31 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #          31      CTTCT CCAGCAGTCT C    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 29 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    #            29    GCAGC CGCGACTGG    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 27 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    #             27   AGTCT CTTCTCC    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 26 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    #              26  TCACA TGCGGG    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 33 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    #         33       NATNG GNATNAAYCA YGG    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 25 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    #               25 ARTTN GACAT    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 28 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    #             28   ARGTN TTYGAYAC    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 26 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    #              26  CCNGG RAANGG    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    #21                RTCNA C    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    #                 22AGNA TG    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    #                23GCCAT NGC    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 17 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    #   17             TG    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    #21                ATCCT C    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 649 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: None    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    #Met Cys Pro Phe Leu Lysln Gln Ser Arg Ala    #                 15    #Ala Thr Ala Thr Arg Prohr Leu Arg Ser Leu    #             30    #Asn Leu Gln Arg Ile Alaly Gly Thr Met Thr    #         45    #Ala Val Gln Ser Ala Arget Ser Lys Ala Leu    #     60    #Ala Ala Gly Val Pro Glyrg Phe Thr Ser Ser    # 80    #Gly Ser Pro Gly Lys Argro Lys Pro Thr Arg    #                 95    #Asn Met Ser Thr Glu Phely Gly Asn Gly Ala    #            110    #Leu Ser Asn Ala Thr Argln Ile His Pro Gly    #        125    #Gly Pro Thr Pro Arg Alaer Ala Thr Val Ser    #    140    #Phe Tyr Asn Ala Glu Leuhe Asp Tyr Asp Ala    #160    #Tyr Phe Asn Asn Ile Asnsp Lys Ser Tyr Arg    #                175    #Thr Ala Ser Lys Asp Gluhe Pro Arg Ala His    #            190    #Leu Gly Met Gly Arg Asnys Ser Asn Asp Tyr    #        205    #Leu Asp Thr Tyr Gly Alahr Met His Lys Thr    #    220    #His Asn Gln His Ala Valrg Asn Ile Ser Gly    #240    #Gly Lys Glu Ala Ala Leueu Ala Lys Leu His    #                255    #Ala Thr Leu Ala Thr Leuhe Val Ala Asn Asp    #            270    #Ser Asp Ser Leu Asn Hissp Cys Val Ile Leu    #        285    #Gly Arg Lys Lys Met Vally Ile Arg His Ser    #    300    #Thr Lys Leu Ala Ser Leueu Val Asp Leu Glu    #320    #Glu Ser Val Tyr Ser Metys Ile Ile Ala Phe    #                335    #Cys Asp Leu Ala Asp Lysro Ile Glu Ala Ile    #            350    #His Ala Val Gly Met Tyrhe Leu Asp Glu Val    #        365    #Leu Asp Tyr Glu Ile Tyrly Val Ala Glu His    #    380    #Thr Lys Gly Thr Val Metla Asn Pro Leu Ser    #400    #Gly Lys Ala Tyr Gly Cysle Thr Gly Thr Leu    #                415    #Leu Val Asp Thr Ile Argla Gly Ser Ala Ala    #            430    #Ser Leu Pro Pro Ala Thrhe Ile Phe Thr Thr    #        445    #Gln Ala Arg His Gln Glnhr Ala Ile Arg Tyr    #    460    #Ala Val Lys Gln Ser Pheln Leu His Thr Arg    #480    #Pro Ser His Ile Val Proro Val Ile Pro Asn    #                495    #Gln Ala Ser Asp Lys Leula Glu Leu Ala Lys    #            510    #Ile Asn Tyr Pro Thr Valle Tyr Val Gln Ala    #        525    #Pro Thr Pro Gly His Thrrg Leu Arg Ile Thr    #    540    #Val Asn Thr Val Trp Asnis Leu Val Glu Ala    #560    #Lys Ala Met Gly Gly Pherg Ala Ser Asp Trp    #                575    #Glu Asn Gln Pro Ile Trplu Ala Ala Glu Leu    #            590    #Glu Thr Leu Glu Ala Alasn Met Arg Pro Asp    #        605    #Gly Met Lys Ala Gly Glyln Ala Ala Val Pro    #    620    #Ala Asn Pro Ile Gly Alaal Gly Ser Ile Ala    #640    -  Ser Ile Pro Val Ala Ala Ala Ala Glx                     645    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 548 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: None    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    #Asn Ser Ser Ala Ala Valhe Ala Arg Phe Gly    #                 15    #Ala Pro His Ala Lys Asneu Ser Thr Thr Ala    #             30    #Ala Ala Ala Thr Ala Thrhr Gly Ala Gly Ala    #         45    #Ala Ala Ala Ala Asn Hisla Ala Ala Ala Ala    #     60    #Gly Leu Ile Asp Ser Gluly Phe Asp Tyr Glu    # 80    #Arg Tyr Phe Asn Asn Ileeu Asp Lys Ser Tyr    #                 95    #His Arg Gln Arg Glu Alalu Phe Pro Leu Ala    #            110    #Tyr Leu Ala Leu Ser Lysrp Cys Ser Asn Asp    #        125    #Thr Ile Asp Lys Tyr Glysp Ala Met His Lys    #    140    #Gly His Asn Ile Pro Thrhr Arg Asn Ile Ala    #160    #His Lys Lys Glu Gly Alalu Leu Ala Thr Leu    #                175    #Asp Ala Val Leu Ser Leuys Tyr Val Ala Asn    #            190    #Phe Ser Asp Glu Leu Asnys Asp Leu Val Ile    #        205    #Ala Asn Val Lys Lys Hisal Gly Ile Lys His    #    220    #Glu Gln Leu Leu Gln Sersp Leu Asn Glu Leu    #240    #Phe Glu Ser Val Tyr Serro Lys Leu Ile Ala    #                255    #Ile Cys Asp Leu Ala Aspla Asp Ile Glu Lys    #            270    #Val His Ala Val Gly Leuhr Phe Leu Asp Glu    #        285    #His Cys Asp Phe Glu Serla Gly Val Ala Glu    #    300    #Thr Asn Asp Lys Gly Glyle Ala Thr Pro Lys    #320    #Ile Thr Gly Thr Leu Glysp Arg Val Asp Met    #                335    #Ala Ala Ser Arg Lys Leual Gly Gly Tyr Val    #            350    #Phe Ile Phe Thr Thr Threr Phe Ala Pro Gly    #        365    #Ala Ala Ile Arg Tyr Glnet Ala Gly Ala Thr    #    380    #Gln Lys His Thr Met Tyreu Arg Thr Ser Gln    #400    #Pro Val Ile Pro Asn Prois Glu Leu Gly Ile    #                415    #Ala Asp Leu Ala Lys Glnal Leu Ile Gly Asn    #            430    #Ile Tyr Val Gln Ala Ilele Asn Lys His Gln    #        445    #Arg Leu Arg Ile Thr Prola Arg Gly Thr Glu    #    460    #Ile Leu Ile Asn Ala Valsn Asp Leu Ser Asp    #480    #Arg Val Arg Asp Trp Glulu Leu Gln Leu Pro    #                495    #Ser Gly Phe Val Glu Glueu Gly Val Gly Glu    #            510    #Leu Thr Asn Asp Asp Leuer Ser Gln Leu Ser    #        525    #Gln Leu Glu Val Ser Sersp Pro Ile Val Lys    #    540    -  Gly Ile Lys Gln     545    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 587 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: None    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    #Cys Pro Val Leu Ala Arget Leu Leu Gln Cys    #                 15    #Lys Thr His Gln Phe Leueu Gly Lys Val Val    #             30    #Thr Gln Gly Pro Asn Cysys Pro Ile Leu Ala    #         45    #Gly Gly Asp Ser Pro Serys Ala Thr Lys Ala    #     60    #Ser Glu Leu Gln Asp Glyys Pro Phe Met Leu    # 80    #Glu Val Gln Glu Asp Valln Lys Ala Ala Pro    #                 95    #Leu Val Ser Val Ser Leusp Leu Pro Ser Ser    #            110    #Glu Gln Ile Ser Gly Lysly Pro Gln Glu Gln    #        125    #Gly Asn Tyr Val Phe Serln Asn Asn Met Pro    #    140    #Glu Lys Lys Gln Asp Hisrg Asp Lys Ile Met    #160    #Trp Ala Asp Ala Tyr Proys Thr Val Asn Arg    #                175    #Ala Ser Lys Asp Val Serhe Glu Ala Ser Val    #            190    #Ser Arg His Pro Gln Valsp Tyr Leu Gly Met    #        205    #His Gly Ala Gly Ala Glylu Thr Leu Gln Arg    #    220    #Phe His Val Glu Leu Gluer Gly Thr Ser Lys    #240    #Ser Ala Leu Leu Phe Sereu His Gln Lys Asp    #                255    #Phe Thr Leu Ala Lys Ilesn Asp Ser Thr Leu    #            270    #Gly Asn His Ala Ser Metle Tyr Ser Asp Ala    #        285    #Lys Phe Val Phe Arg Hissn Ser Gly Ala Ala    #    300    #Glu Lys Ser Asn Pro Lyseu Lys Lys Leu Leu    #320    #His Ser Met Asp Gly Alala Phe Glu Thr Val    #                335    #Ser His Gln Tyr Gly Alalu Leu Cys Asp Val    #            350    #Gly Leu Tyr Gly Ser Arglu Val His Ala Val    #        365    #Met His Lys Ile Asp Ilelu Arg Asp Gly Ile    #    380    #Cys Val Gly Gly Tyr Ilely Lys Ala Phe Gly    #400    #Arg Ser Tyr Ala Ala Glyeu Val Asp Met Val    #                415    #Val Leu Ser Gly Ala Leuer Leu Pro Pro Met    #            430    #Gly Gln Ala Leu Arg Argeu Lys Gly Glu Glu    #        445    #Gln Leu Leu Met Asp Argal Lys His Met Arg    #    460    #Ile Ile Pro Ile Arg Valro Cys Pro Ser His    #480    #Asp Leu Leu Leu Ser Lyssn Ser Lys Leu Cys    #                495    #Pro Thr Val Pro Arg Glyln Ala Ile Asn Tyr    #            510    #His His Ser Pro Gln Meteu Ala Pro Ser Pro    #        525    #Ala Trp Thr Ala Val Glylu Lys Leu Leu Leu    #    540    #Cys Asn Phe Cys Arg Argal Ser Val Ala Ala    #560    #Glu Arg Ser Tyr Phe Glyeu Met Ser Glu Trp    #                575    #Alasn Met Gly Pro Gln Tyr Val Thr Thr Tyr    #            585    __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid fragment comprising anucleic acid sequence which encodes a 5-aminolevulinic acid synthaseobtained from an Aspergillus oryzae strain.
 2. A nucleic acid fragmentaccording to claim 1, wherein the nucleic acid sequence encodes a5-aminolevulinic acid synthase obtained from Aspergillus oryzae IFO4177.
 3. A nucleic acid fragment according to claim 2, wherein thenucleic acid sequence is set forth in SEQ ID NO:1.
 4. A nucleic acidfragment according to claim 1, which is capable of hybridizing underconditions of high stringency with a probe which hybridizes with thenucleic acid sequence set forth in SEQ ID NO:1 under the conditions ofhigh stringency.
 5. A nucleic acid construct comprising a nucleic acidfragment of claim 1 operably linked to regulatory regions capable ofdirecting the expression of the 5-aminolevulinic acid synthase in asuitable expression host.
 6. A nucleic acid construct according to claim5, wherein the nucleic acid sequence encodes the 5-aminolevulinic acidsynthase of Aspergillus oryzae IFO
 4177. 7. A nucleic acid constructaccording to claim 5, wherein the nucleic acid sequence is capable ofhybridizing under conditions of high stringency with a probe whichhybridizes with the nucleic acid sequence set forth in SEQ ID NO:1 underthe conditions of high stringency.
 8. A recombinant vector comprising anucleic acid construct of claim
 5. 9. A vector according to claim 8,wherein the nucleic acid sequence is operably linked to a promotersequence.
 10. A vector according to claim 9, further comprising atranscription termination signal.
 11. A vector according to claim 9,further comprising a selectable marker.
 12. A recombinant host cellcomprising the nucleic acid construct of claim
 5. 13. A host cellaccording to claim 12, wherein the nucleic acid construct is containedon a vector.
 14. A host cell according to claim 12, wherein the hostcell is a bacterial cell.
 15. A host cell according to claim 12, whereinthe host cell is a fungal cell.
 16. A host cell according to claim 15,wherein the fungal cell is a filamentous fungal cell.
 17. The host cellaccording to claim 16, wherein the filamentous fungal cell is a cell ofa species of Acremonium, Aspergillus, Fusarium, Humicola,Myceliophthora, Mucor, Neurospora, Penicillium, Thielavia,Tolypocladium, or Trichoderma.
 18. A host cell according to claim 17,wherein the filamentous fungal cell is a Fusarium cell.
 19. A host cellaccording to claim 17, wherein the filamentous fingal cell is anAspergillus cell.
 20. A host cell according to claim 15, wherein thefungal cell is a yeast cell.
 21. A host cell according to claim 20,wherein the yeast cell is a strain of Saccharonyces orSchizosaccharomyces.
 22. A host cell according to claim 12, wherein thenucleic acid construct is integrated into the host cell genome.
 23. Amethod for producing a 5-aminolevulinic acid synthase obtained from anAspergillus oryzae comprising (a) cultivating a host cell comprising anucleic acid construct comprising a nucleic acid sequence encoding the5-aminolevulinic acid synthase under conditions conducive to expressionof the 5-aminolevulinic acid synthase; and (b) recovering the5-aminolevulinic acid synthase.